The present invention relates to oxazine derivatives, methods for their production and their application as fluorescent dyes for labelling biological and non-biological materials in the red spectral range.
Oxazine-based dyes are widely used. They are frequently found in the textile industry for dyeing materials of various kinds. Thus, EP-B-0 603 129 describes oxazine dyes which each have exo-amino groups at the benzene rings which can be substituted. The barely soluble salts of these basic oxazine dyes are suitable for dyeing or printing natural fibres and completely synthetic fibres.
U.S. Pat. No. 5,656,759 discloses a cationic oxazine dye which also has two exo-amino groups which can be substituted. The oxazine dyes which can be made hydrophobic by substitution of inorganic anions are especially used in the ink layer of an ink ribbon for thermal transfer printing.
EP-A-0 747 447 discloses oxazine dyes used as fluorescence markers in connection with biological molecules. The absorption range of these conjugate forms is situated at 645–700 nm and corresponds to the red spectral range. The linkable oxazine derivatives are at least tetracyclic systems in which at least one of the two exocyclic nitrogens is integrated in the ring structure. As a result of the complex polycyclic basic structure, however, the expenditure for synthesising these dyes is very high.
Especially for high-throughput screening using confocal fluorescence spectroscopy there is a need for dyes which have a high fluorescence quantum yield, high solubility in water, high solubility in organic solvents, high chemical stability, high photostability and the capacity to bind to biological and non-biological materials or molecules.
As a result of the cheap access to red-emitting laser light sources and the low background fluorescence (autofluorescence) of biological material excited in the red compared with the energy-richer exciting wavelengths, the use of fluorescence dyes excitable in the red, including among others the oxazine dyes, is acquiring increasing importance.
As a result of conventional labelling methods, especially protein labelling methods, for example, using fluorescent detection markers, multiple reactive groups of these molecules are being targeted so that the labelling reaction results in a statistical labelling i.e., some proteins possess no labels, others possess one, two or more. In highly sensitive analytical techniques the heterogeneous analyte mixture leads to undesirable heterogeneous signal distributions which falsify the measurement results or make them completely unusable. In addition, as a result of interaction between the individual labellings in multiply labelled proteins, for example, there is a weakening of the signal.
The object of the present invention is to provide fluorescent dyes which avoid the aforesaid disadvantages of known fluorescent dyes and exhibit said positive properties. It is a further object of the invention to provide methods for producing these fluorescent dyes and a method for labelling material or molecules using these dyes. The object especially consists in providing red-excitable fluorescent dyes which are highly soluble in water, wherein the photochemical properties are retained. Furthermore, the dye should be activated in a fashion such that it is made capable of linking to biological and non-biological materials or molecules without there being any negative interaction with the materials or molecules to be labelled.